Novel femtosecond light source aids biosensing

An ideal laser for fluorescence lifetime
systems should have a repetition rate of several megahertz, a pulse duration much
shorter than the fluorescence decay of the dye and the appropriate operating wavelength.
That combination has proved challenging to achieve.

Now researchers at the University of Toronto and
at the University of Toronto at Mississauga, both in Ontario, Canada, have developed
a femtosecond laser — based on an ytterbium-doped potassium-gadolinium-tungstate
crystal — that fits these requirements. The Yb:KGW laser could act as the
source in tools for clinical diagnostics, environmental or food safety monitoring,
biowarfare agent detection, veterinary testing or other applications.

Researchers have designed an Yb:KGW laser. An infrared image of its
laser head is shown.
“The ultimate goal is to use
this technique as a platform for rapid, reusable, sensitive and selective nucleic
acid diagnostics,” said Paul A.E. Piunno of the University of Toronto at Mississauga.

Piunno and Sergei Musikhin of the Mississauga
campus developed optical DNA biosensors in conjunction with the laser. The team
also included postdoctoral researcher Arkady Major of the University of Toronto,
who developed and characterized the laser system.

According to Major, the goal was to
build a reliable and cost-effective ultrashort pulse laser that could provide green
illumination. Those characteristics would enable the construction of a DNA biosensor
based on the fluorescent label ethidium bromide, or any other dye that also absorbs
in the green. The fluorophore has a fluorescence lifetime of ~1.5 to 2.0 ns
when free in solution, but the lifetime climbs to ~21 ns when the dye is bound
to double-stranded nucleic acid structures. That difference is easy to detect and
acts as a signal.

The Yb:KGW laser could be useful in biosensing. The design of the sensor cartridge is
shown above and the layout of the experiment below.
However, widely available lasers do
not meet the illumination requirements. Dye lasers have repetition rates of a few
hertz and nanosecond pulse durations, so they are too slow and the pulses too long
for rapid and accurate fluorescence lifetime measurements. On the other hand, femtosecond
lasers have short enough pulses, but they typically operate with repetition rates
of 100 MHz. Rapidly repeating pulses can excite the dye and other molecules before
they have a chance to relax to a ground state, thereby potentially throwing off
the fluorescence lifetime determination.

Because ultrashort pulses can be frequency-doubled
using a nonlinear crystal, the researchers wanted a laser with a fundamental wavelength
of about 1 μm. They used Yb:KGW instead of perhaps better known alternatives
because it is pumpable by telecom-grade laser diodes operating at 970 to 980 nm.
Moreover, Yb:KGW supports 100-fs pulses at a wavelength of just over 1 μm.

The crystal selection led to a laser
with significant manufacturing advantages. “Its design is very straightforward
and takes advantage of commercially available long-lifetime telecom components and
off-the-shelf high-quality laser optics. The construction was simple,” Major
said.

To decrease the repetition rate, the
researchers extended the laser cavity, a technique that had the advantage of less
complexity and lower cost than other methods.

To do this, they constructed two optical
telescopes, using mirrors to fold their length. They extended the cavity up to 16
m per round-trip, resulting in a 15-MHz repetition rate. The 67 ns between two consecutive
pulses were longer than the 20-ns fluorescence lifetime of bound ethidium bromide.

The pulses were about 180 fs in duration
and centered around 1042 nm. The scientists sent them into a nonlinear crystal,
producing a second harmonic at ~520 nm, which is the excitation peak of the
fluorophore. The work is detailed in the June 12 issue of Optics Express.

To demonstrate in a preliminary feasibility
study that the laser could be used as a source for optical detection of DNA, the
researchers utilized a setup with focusing and collecting optics, a Hamamatsu photomultiplier
and a Becker & Hickl single-photon counting device.

The frequency-doubled pulses entered
a solution containing ethidium bromide and double-stranded 20-mer oligonucleotides
in one case, and just the fluorophore in the other. The fluorescence lifetime in
the bound case, the one with the DNA, was ~20 ns, while in the other case,
the lifetime was ~2 ns.

With the feasibility shown, the researchers
are working on an optical DNA sensor based on total internal reflection. In these
studies, the sensor consists of a prism whose base has been functionalized with
a DNA sequence; for example, one that could act as a diagnostic for a degenerative
disease.

The sample DNA, which in the demonstration
would be complementary to the oligonucleotides attached to the prism, would flow
past this surface in a microfluidic device, along with an equivalent concentration
of ethidium bromide. The change in fluorescence lifetime indicates whether the fluorophore
is bound by the DNA or not, and that binding also can be switched off by heating
the fluid using a temperature-controlled plate in the device. This work is ongoing.

The group also is working on further
improvements in which more miniaturization of the sensor elements is one of the
goals, but Piunno reported that this was not the only goal. “A microfluidic
construct is being explored while the optics of the system are being reworked so
as to provide for higher-sensitivity detection,” he said.

The laser also could be used in a variety
of other applications. Major noted that the laser emits at 1 μm, which is longer
than the 800 μm of Ti:sapphire femtosecond lasers. That longer wavelength reduces
scattering by and increases penetration depth of biological tissue. It also produces
a third harmonic that is not in the deep ultraviolet.

“[The Yb:KGW] is a good alternative
to commonly used Ti:sapphire lasers,” Major said.